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Peptide microarray-based fluorescence assay for quantitatively monitoring the tumor-associated matrix metalloproteinase-2 activity
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Peptide microarray-based fluorescence assay for quantitatively monitoring the tumor-associated matrix metalloproteinase-2 activity Minghong Jiana,b, Min Sua, Jiaxue Gaoa,c, Zhenxin Wanga,b,* a
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, PR China School of Applied Chemistry and Engineering, University of Science and Technology of China, Jinzhai Road Baohe District, Hefei, Anhui, 230026, PR China c University of Chinese Academy of Sciences, Beijing, 100049, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Matrix metalloproteinase-2 Peptide microarray-based fluorescence assay serum Human osteosarcoma
Matrix metalloproteinases (MMPs) are important biomarkers of various tumors. Herein, a peptide microarraybased fluorescence assay is developed for quantitatively profiling of MMP-2 activity in different matrices through the binding of the immobilized biotinylated peptides on the microarray with the fluorescein isothiocyanate (FITC) modified neutravidins. In the presence of MMP-2, the biotin moiety is released from microarray by enzymatic cleavage of peptide substrate, resulting in the decrease of fluorescence signal. The change of fluorescence intensity is correlated with MMP-2 activity. The detection limit down to 14 pg mL−1 in buffer solution is obtained by a selected peptide substrate for MMP-2 from eleven peptide substrate candidates. The cellular secreted MMP-2 activity levels of different living cells are further successfully quantitatively evaluated by the selected peptide substrate. Using mouse-bearing MG-63 human osteosarcoma as a model system, we also demonstrate that the activity of MMP-2 in serum is closely related with the cancer progression.
1. Introduction Matrix metalloproteinases (MMPs) are a kind of proteolytic enzymes with Zn2+ and Ca2+, which degrade the extracellular matrix components (ECM) and play crucial roles in physiological and pathological processes, such as normal tissue remodeling, embryogenesis, angiogenesis, wound healing, arthritis, atheroma, tissue ulceration, and cancer [1–5]. In particular, MMPs are demonstrated as useful diagnostic and prognostic biomarkers for cancer because the up-regulation of various MMPs, including MMP-1, -2, -3, -7, -9, -13, -14 are positively related with tumor progression in both primary tumors and metastases [6–10]. Due to their importance in physiological and pathological processes, many approaches/methods have been developed for detection of MMPs, including gelatinase zymography [11,12], enzyme-linked immunosorbent assays (ELISA) [13], surface plasmon resonance (SPR) assays [14,15], electrochemical biosensors [16], and fluorescence resonance energy transfer (FRET) assays [17,18]. Although these methods have reasonable accuracy and sensitivity, most of them require relative large sample volumes and cannot detect MMP activities in the high throughput format. Moreover, targeting MMP specifically is still a challenge because the active sites of MMP family members are
structurally similar [19,20]. Screening of specific peptide substrates for MMPs is beneficial for the detection MMPs activities precisely. With the advantage of massively parallel detection of a large number of different targets on a small scale, microarray has been considered as a revolutionized technology for biological detection and monitoring since it was invented in the early 1990s [21–23]. For example, peptide microarrays are extensively employed to screening antigens in practical samples and study the functionalities and inhibitions of enzymes [24–27]. Very recently, we have developed a series of peptide microarray-based fluorescence assay for profiling multiplexed MMPs activities in cell lysates and clinical thyroid tissues [28,29]. It is found that the different clinical samples exhibit different expression patterns of MMP activities. Currently, extracts of pathological tissues are normally used for analysis of tumor-associated MMPs activities [30–32]. There are few examples of monitoring MMPs activities in serum samples. Although tissue testing is a golden standard for clinical diagnosis of tumor, it is difficult to obtain tissue samples under certain conditions, such as tumorlets and brain metastasis. In addition, tissue biopsies may increase the risk of tumor metastasis. Compared to tissue biopsy, blood biopsy offers the possibility with minimize invasiveness to characterize the characteristic molecular information on tumor and monitor the
⁎ Corresponding author at: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, PR China. E-mail address: [email protected] (Z. Wang).
https://doi.org/10.1016/j.snb.2019.127320 Received 8 August 2019; Received in revised form 9 October 2019; Accepted 21 October 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Minghong Jian, et al., Sensors & Actuators: B. Chemical, https://doi.org/10.1016/j.snb.2019.127320
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2.3. Determination of MMP-2 activity in buffer
progression of cancer through real-time dynamic monitoring the expression levels of biomarkers in the blood samples of patients [33]. Therefore, MMP activity-based liquid biopsy in the blood draw has great potential in the routine diagnosis and prognosis of tumors. Herein, we have developed a peptide microarray-based fluorescence assay for profiling MMP-2 activity with high sensitivity in various biological samples, including cell culture medium, tissue extract and serum by a selected peptide substrate. Using mouse-bearing MG-63 human osteosarcoma as a typical model, the as-proposed peptide microarray-based fluorescence assay is used to robustly quantify the differential activities of MMP-2 in the serum from mice with different cancer progression. It is demonstrated that the activity of MMP-2 is associated with tumor progression.
The 10 nM proMMP-2 and 100 nM proMMP-7 were activated with 1 mM APMA in TCNB buffer at 37 °C for 1 h and 2 h, respectively. Subsequently, 30 μL various concentrations of pure MMP-2 and MMP-7 in TCNB buffer were added to each subarray and incubated at 37 °C for 4 h. The subarray treated with TCNB buffer was used as a blank control. Then, the slides were washed with washing buffer 2 for 5 min (3 × 30 mL), and Milli-Q water for 3 min (3 × 30 mL), respectively. After dried by centrifugation, 30 μL 3 μM Neutravidin-FITC in probe buffer (pH 7.5, 50 mM PB, 0.15 M NaCl, 0.1% Tween-20 (v/v) and 1% BSA (w/v)) were added to each subarray and incubated at 30 °C under dark for 1 h. After PTFE masks were removed, the glass slides were washed with PBS (pH 7.5, 0.05 M PB, 0.15 M NaCl, supplemented with 0.1% Tween-20 (v/v)) for 5 min (3 × 30 mL), and Milli-Q water for 3 min (3 × 30 mL), and dried via centrifugation before scanning, respectively. To investigate the specificity of as-proposed method for detection of MMP-2, 30 μL different enzymes (caspase 3, cathepsin B, pronase, λexo and proteinase K) in reaction buffer were added to each subarray and incubated for 4 h, respectively. The subarrays were then treated as previously described.
2. Experimental section 2.1. Reagents Recombinant human matrix metalloproteinase 2 (proMMP-2) and caspase 3 were purchased from Sino Biological Inc. (Beijing, China). Recombinant human matrix metalloproteinase 7 (proMMP-7) and cathepsin B were purchased from R&D Systems Inc. (Minneapolis, USA). Pronase from streptomyces griseus and proteinase K were purchased from Sigma-Aldrich Co. (St Louis, USA). λ exonuclease (λexo) was acquired from New England Biolabs Ltd. (Hitchin, UK). Neutravidin-FITC was acquired from Thermo Fisher Scientific Co., Ltd. (China). (4-aminophenyl) mercuric acetate (APMA) was obtained by GenMed Medical Science and Technology Ltd. (Shanghai, China). The peptide substrates (as shown in Table S1) were synthesized by Synpeptide Co., Ltd. (Shanghai, China). Bovine Serum Albumin (BSA) was acquired from GEN-VIEW Scientific Inc. (USA). Dulbecco modified eagle medium (DMEM) were supplied by Beijing Dingguo Biotechnology Ltd. (Beijing, China). McCoy’s 5A culture medium was purchased from Jiangsu KeyGEN BioTECH Co. Ltd. (Jiangsu, China). Fetal bovine serum (FBS) and trypsin-EDTA cell detaching kit were purchased from Gibco Co. (New York, USA). Human serum was obtained from First Hospital of Jilin University (Changchun, China). Gelatin-sepharose 4B was received from Solarbio Science & Technology Co., Ltd. (Beijing, China). MMP-2 activity quantification kit and Bradford protein concentration quantification kit were acquired from Shanghai Harling Biotechnology Co., Ltd. (Beijing, China). 3D aldehyde glass slides and polytetrafluoroethylene (PTFE) masks were supplied by CapitalBio Ltd. (Beijing, China). NCM460, HepG2, SW620, HCT116, HeLa and MG-63 cells were acquired from Chinese Academy of Sciences Cell Bank (Shanghai, China). All other reagents were of analytical grade. Milli-Q water (18.2 MΩ⋅cm) was used in all experiments.
2.4. Data acquisition and analysis The fluorescence signals were obtained with a LuxScan-10 K fluorescence microarray scanner (CapitalBio Ltd., Beijing, China), and background fluorescence signals were subtracted. The average value and standard deviation of each sample was calculated from six repeated signal spots. The relative change of fluorescence intensity (ΔFR) was used to represent the MMP-2 activity, which was calculated by the following formula: ΔFR = (F0–F)/F0 × 100%, where F0 and F indicate the average fluorescence signal before and after MMP-2 cleavage, respectively. The detection limit was estimated as 3 times the standard deviation of ΔFR of control samples. 2.5. Detection of cellular MMPs activities NCM460, HepG2, SW620, HCT116, HeLa and MG-63 cells were seeded in 48-well plates at a density of 5000 cells per well and grew in complete medium (fresh culture medium containing 10% fetal bovine serum (FBS) and 100 U mL−1 penicillin-streptomycin) in humidified air with CO2 at 37 °C for 24 h. NCM460, SW620 and HCT116 were cultured with McCoy’s 5A. HeLa, MG-63, and HepG2 were cultured with DMEM. The cells were washed with PBS, and incubated with fresh serum-free medium for 12 h. The culture medium was discharged. After washed with PBS, the starved cells were incubated with serum-free medium for another 24 h. Subsequently, the culture medium was collected and centrifuged (1000 rpm) at 4 °C for 10 min. Finally, the obtained supernatant was adjusted to pH 7.5 and 30 μL samples were added to each subarray for MMP-2 activity detection. The subsequent experimental procedure was as same as described to detect MMP-2 activity in buffer. For comparison, the cellular secreted MMP-2 activity in concentrated (10 times) sample was also detected by the commercial MMP-2 activity quantification kit.
2.2. Fabrication of peptide microarray The different concentrations of peptide substrates in spotting buffer (pH 8.5, 0.3 M PB, 0.2 M NaCl, containing 20 μg mL−1 BSA and 35% (v/ v) glycerol) were spotted on commercial 3D aldehyde glass slides with a SmartArrayer 136 system using a standard contact printing procedure (Capital Bio, Beijing, China). After an overnight incubation under vacuum at 30 °C, the peptide microarrays were washed with washing buffer 1 (pH 7.5, 50 mM PB, 1% (w/v) BSA) for 10 min twice, and then incubated in blocking buffer (pH 7.5, 50 mM PB, 0.15 M NaCl, 1% (w/ v) BSA and 1% (v/v) ethanolamine) at 30 °C for 1 h to block the residual aldehyde groups on the slides. After the blocking process, the slides were successively washed with washing buffer 2 (pH 7.5, 20 mM Tris, 0.15 M NaCl with 0.1% Triton X-100) for 10 min (2 × 30 mL), and then TCNB buffer (pH 7.5, 50 mM Tris, 10 mM CaCl2, 150 mM NaCl and 0.05% Brij-35) for 10 min (2 × 30 mL), respectively. Finally, the peptide microarray was dried by centrifugation at 2000 rpm for 1 min, and divided into 12 individual subarrays using a PTFE mask for further study of the MMPs activities.
2.6. Detection of MMP-2 activity in serum and tissue All the animal experiments were approved by the Regional Ethics Committee of Jilin University, and complied with the guidelines for care and use of laboratory animals. Male NOD/SCID mice (18–20 g bodyweight) were obtained from Beijing HFK Bioscience Co., Ltd. (Beijing, China), which were housed in a 12 h light/dark cycle with a controlled temperature and humidity, and fed with a chow diet and drinking tap water. After acclimatizing to the environment for one week, each NOD/SCID mouse was inoculated subcutaneously with MG2
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63 cells (1.0 × 106 cell in 100 μL PBS). When the tumor grew to a certain volume, the mice were anesthetized by isoflurane (Shenzhen RWD Life Technology Co., Ltd.). The tumors and bloods of mice were collected. The leg muscles and bloods of healthy NOD/SCID mice were collected as control samples. After standing at the room temperature for 1 h, the blood samples of NOD/SCID mice were centrifuged (1500 rpm) at 4 °C for 10 min. The serum was then collected. MMPs of tissue samples were extracted and purified according to the previously reported procedure [29]. Generally, the tissues (30 mg) were cut into small pieces with scissors and homogenized with 400 μL working buffer (pH 7.6, 50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, 0.05% (w/v) Brij35, and 0.02% (w/v) NaN3) containing 1% (v/v) Triton X-100 in 1 mL Teflon-glass homogenizer on ice. The total protein content in 10 μL homogenate was calculated using the bicinchoninic acid (BCA) method. The homogenate supernatant was collected by centrifugation (12,000 rpm) at 4 °C for 15 min, and then incubated with 50 μL gelatinsepharose 4B under constant shaking (100 rpm) for 1 h. The gelatinsepharose 4B was pretreated by working buffer (3 × 100 μL). After centrifuged at 7000 rpm for 5 min, the gelatin-sepharose pellet was resuspended in 200 μL working buffer and centrifuged again. Then, the pellet was incubated with 50 μL elusion buffer (working buffer containing 10% (v/v) DMSO) under constant shaking (100 rpm) for 30 min. The mixture was centrifuged (7000 rpm) at 4 °C for 5 min, and the supernatant was diluted to suitable volume for detecting MMPs activities. The activities of MMP-2 in serum and/or tissue extracts were detected by both the peptide microarray-based fluorescence assay and commercial MMP-2 activity quantification kit as previously described, respectively.
Fig. 1. Fluorescence images (a and b) and corresponding data analysis (c) of different peptide substrates (0.1 mg mL−1) cleaved by MMP-2 and MMP-7, respectively. The concentrations of MMP-2 and MMP-7 are 250 ng mL−1 and 100 ng mL−1, respectively.
3. Results and discussion
from the literature reported different preferred MMP-2 and MMP-7 cleavage motifs because MMP-2 and MMP-7 are normally overexpressed in the various tumors [5,9,35]. The reactions of immobilized peptides on microarray with MMPs were carried out under our previously optimized experimental conditions [29,36]. As shown in Fig. 1, there are obvious differences in the enzymatic cleavage efficiencies of S0, S3, S6 and S7 by MMP-2 and MMP-7. In particular, S0 shows much higher (5.5 times) MMP-2 cleavage efficiency than MMP-7 cleavage efficiency. The result indicates that S0 has relative high specificity for MMP-2. Therefore, S0 was employed to evaluate the activity of MMP-2 in the following experiments.
3.1. Screening the specific peptide substrate for MMP-2 Scheme 1 illustrates the detection of MMP activity using peptide microarray-based fluorescence assay. Briefly, the biotin-modified peptides were immobilized on the aldehyde 3D glass slides via forming a stable five-membered-ring thiazolidine between the N-terminals of peptide substrates and aldehyde groups on the slides [34]. NeutravidinFITC can specifically reacted with the biotin group at the C-terminal of peptide substrate to produce strong fluorescence signal. When MMPs were introduced, the peptide substrates were enzymatically cleaved and the peptide fragments containing biotin were released from the slides, resulting in the decrease of the fluorescence signal. The activities of MMPs can be expressed by fluorescence intensity changes on the microarrays. For improving the detection performance of MMPs, eleven biotinmodified peptides (named as S0 to S10) containing MMP recognition sequences were randomly designed to select the substrates of MMP-2 with relatively high specificity. The recognition sequences are derived
3.2. Determination of MMP-2 activity in buffer The effect of the surface densities of S0 on the enzymatic cleavage efficiency was firstly investigated. Figure S1 illustrates that the highest enzymatic cleavage efficiency was obtained when the concentration of S0 was 0.1 mg mL−1 in the spotting solution. In the following studies, 0.1 mg mL−1 was chosen as the optimized concentration of S0 to
Scheme 1. The schematic representation of as-proposed peptide microarray-based fluorescence assay for determining MMP-2 activity. The illustration is not drawn to scale. 3
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detection are summarized in Table 1. The results are comparable to or better than those reported in the literatures [14,18,38]. For testing its specificity for MMP-2 detection, the as-proposed peptide microarray-based fluorescence assay was employed to react with different enzymes including caspase 3, cathepsin B, pronase, λexo and proteinase K. As shown in Figure S2, those enzymes show negligible cleavage efficiency on S0, indicating that the as-proposed peptide microarray-based fluorescence assay has excellent specificity for detecting MMP-2 activity. 3.3. Detection of cellular secreted MMP-2 activities To further study its performance, the activity of cellular secreted MMP-2 was detected by the peptide microarray-based fluorescence assay. Herein, six cell lines were selected including one normal cell (human colon epithelial cell (NCM460)), five cancer cells (human cervical adenocarcinoma cell (HeLa), two kinds of human colorectal cancer cells (HCT116 and SW620), human hepatoma cell (HepG2) and human osteosarcoma cell (MG-63)). All cells were seeded with an optimal density of 5000 cells per well [29]. The activity of cellular secreted MMP-2 in culture medium was detected by the peptide microarray-based fluorescence assay. The activity level of cellular secreted MMP-2 exhibits significant difference among different cell lines (as shown in Fig. 3). For instance, the activities of cellular secreted MMP-2 by tumor cells are higher than that of the normal cell (NCM460). Although both SW620 and HCT116 are derived from colorectal cancer, the activity of cellular secreted MMP-2 by SW620 is higher than that of HCT116. In addition, the activities of secreted MMP-2 by SW620, MG63 and HepG2 are higher than those of HCT116 and HeLa. The result suggest that the MMP activity is associated to pathological characteristics of tumors, such as the degree of tumor malignancy [7,39–41]. The activity levels of cellular secreted MMP-2 by different cells were also determined by commercial MMP-2 activity quantitative determination kit (as shown in the Fig. 3), which are comparable with those obtained from the peptide microarray-based fluorescence assay. Furthermore, the sensitivity of peptide microarray-based fluorescence assay is much higher than that of commercial MMP-2 activity quantitative determination kit. The result demonstrates that the peptide microarray-based fluorescence assay can be used to effectively detect MMP-2 activity in complex biological samples.
Fig. 2. Fluorescence images (a to d) and the performance of as-proposed peptide microarray-based fluorescence assay (e) for determining MMP-2 activity in TCNB buffer (a), DMEM (b), human serum (c) and DMEM supplemented with human serum (d), respectively.
fabricate peptide microarray. Various concentrations of activated MMP2 were prepared for evaluating the performance of as-proposed peptide fluorescence assay. As shown in Fig. 2, ΔFR of S0 is linearly increased with increasing the logarithm of MMP-2 concentration. For testing the practical capability of the assay, the potential interfering substances in biological samples were investigated. The various amounts of activated MMP-2 were mixed with DMEM, 5% human serum in TCNB buffer and/ or DMEM supplemented with 5% human serum. The mixtures were then applied to subarrays. It can be seen from Fig. 2 that the interfering substances have strong effect on the detection sensitivity (slope of calibration curve) of the assay because these biosubstances normally contain small amounts natural MMP inhibitors such as TIMPs (tissue inhibitors of metalloproteinases, e.g., TIMP-1 and TIMP-2) or α2-macroglobulin [35,37]. However, the interfering substances show slight effect on the detection limit and linear range. For comparison, detection sensitivities, the linear ranges, linear regression equations and limits of
3.4. Analysis of MMPs activities in serum and tissue Encouraged by the detection result of cellular secreted MMP-2, the peptide microarray-based fluorescence assay was further used to study the relationship of MMP-2 activity with tumor progression. In this case, a NOD/SCID mouse-bearing MG-63 human osteosarcoma was used as a typical model system because human osteosarcoma has high metastatic characteristics [42]. The tumor tissues and bloods of mice were collected when the tumor grew to a specific size (0 (0 means healthy mouse), 0.5 cm, 0.8 cm or 1.2 cm maximum diameter of tumor, as shown in Fig. 4a). We systematically evaluated the MMP-2 activities of the as-collected tumor tissues and blood samples by the peptide microarray-based fluorescence assay and commercial MMP-2 activity quantitative determination kit. Different dilution volume ratios (1%, 5%, 10%, 50% and 100%) of serum with TCNB buffer were applied to
Table 1 Summary of the performance of as-proposed peptide microarray-based fluorescence assay for determining MMP-2 activity in different matrices. Matrix
Detection sensitivities
linear ranges
Linear regression equations
Limits of detection
TCNB DMEM Human Serum DMEM and Human Serum
9.32438 5.92849 5.80310 4.77283
25 25 25 25
Y = 9.32438X-7.78706 Y = 5.92849X-1.87238 Y = 5.80310X-3.16496 Y = 4.77283X-0.91547
14 pg/mL 19 pg/mL 22 pg/mL 25 pg/mL
pg/mL-500 ng/mL pg/mL-500 ng/mL pg/mL-500 ng/mL pg/mL-250 ng/mL
4
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Fig. 3. Profiling of cellular secreted MMP-2 activity in NCM460, HepG2, SW620, HCT116, HeLa, and MG-63 cell culture medium by peptide microarraybased fluorescence assay and commercial MMP-2 activity quantitative determination kit. The insets are the corresponding fluorescence images. For peptide microarray-based fluorescence assay, the MMP-2 amount was estimated by the calibration curve of MMP-2 in DMEM (as shown in Fig. 2e). The sample amount of commercial kit is 10 times larger than that of peptide microarraybased fluorescence assay.
Fig. 5. Profiling of MMP-2 activity in tissues homogenate obtained from the mice with different stages of MG-63 human osteosarcoma by peptide microarray-based fluorescence assay and commercial MMP-2 activity quantitative determination kit. The insets are the corresponding fluorescence images. For peptide microarray-based fluorescence assay, the MMP-2 amount was estimated by the calibration curve of MMP-2 in DMEM plus human serum (as shown in Fig. 2e). The sample amount of commercial kit is 10 times larger than that of peptide microarray-based fluorescence assay.
the subarrays for investigating the effect of serum concentration on the MMP-2 activity, because the components in serum (e.g., serum albumin and cytokines) may affect the interaction of MMP-2 with the immobilized S0 on microarrays. The MMP-2 activity in serum can be detected as low as 1% diluted serum, while the maximum cleavage efficiency was achieved with 5% diluted serum (as shown in Fig. 4b). Therefore, the 5% diluted serum was used to investigate the relationship of serum MMP-2 activity with tumor progression. Given a reaction solution volume of 30 μL, this means that the MMP-2 activity in 1.5 μL serum can be detected. As expected, the serum MMP-2 activity level is increased with increasing the tumor size (as shown in Fig. 4c). The MMP-2 activity level in tumor tissue was also determined by the peptide microarray-based fluorescence assay. The MMP-2 activity level in tumor tissue is also proportional to the tumor size (as shown in Fig. 5), i.e., the MMP-2 activity is strongly associated with osteosarcoma progression, which is consistent with the previously reported results [43–45]. The consistency of MMP-2 activity in serum and tissue suggests that MMP activity-based liquid biopsy could be used to monitor the progress of tumors. Moreover, the activities of MMP-2 in serum samples and tumor tissues were also detected by commercially available MMP-2 activity quantitative determination kits, and the results are in consistent with those of the peptide microarray-based fluorescence assay. 4. Conclusions In summary, we have proposed a peptide microarray-based fluorescence assay for profiling the MMP-2 activity in different biological samples (i.e., cell lysates, serum and tissue homogenate) with a wide dynamic range and relative high sensitivity. Using MG-63 tumorbearing NOD/SCID as a model, we demonstrate that MMP-2 activity is strongly associated with the tumor progression. Although only MG-63 human osteosarcoma related MMP-2 activity is detected in this proof of principle experiment, this approach offers a possibility for the development of MMP activity-based tumor liquid biopsy in the blood draw.
Fig. 4. Profiling of MMP-2 (b) activity in different concentrations of serum in TCNB buffer obtained from the mice with different stages of MG-63 human osteosarcoma (a) by peptide microarray-based fluorescence assay. Data analysis of MMP-2 activity in 5% serum measured by peptide microarray-based fluorescence assay and commercial MMP-2 activity quantitative determination kits (c). The insets are the corresponding fluorescence images. For peptide microarray-based fluorescence assay, the MMP-2 amount was estimated by the calibration curve of MMP-2 in human serum (as shown in Fig. 2e). The sample amount of commercial kit is 10 times larger than that of peptide microarraybased fluorescence assay.
Declaration of Competing Interest The authors declare no competing financial interest. Acknowledgement The authors would like to thank the National Natural Science Foundation of China (Grant no. 21775145) for financial support. 5
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Appendix A. Supplementary data
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Minghong Jian is currently a PhD student in the University of Science and Technology of China. She is currently working on the detection of enzyme activity in biological samples using peptide microarrays. Min Su completed her PhD degree in 2014 from University of Chinese Academy of Sciences. Her current scientific interest concentrates on the detection of thrombin enzyme. Jiaxue Gao is pursuing PhD degree at the University of Chinese Academy of Sciences. She works on cancer related-miRNA detection in living cells. Zhenxin Wang is a professor at State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, since 2006. He completed his PhD degree in the Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, in 2000. His main research interests concentrate on microarrays, multimodal imaging in vivo and biomaterials.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Peptide microarray-based fluorescence assay for quantitatively monitoring the tumor-associated matrix metalloproteinase-2 activity Minghong Jiana,b, Min Sua, Jiaxue Gaoa,c, Zhenxin Wanga,b,* a
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, PR China School of Applied Chemistry and Engineering, University of Science and Technology of China, Jinzhai Road Baohe District, Hefei, Anhui, 230026, PR China c University of Chinese Academy of Sciences, Beijing, 100049, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Matrix metalloproteinase-2 Peptide microarray-based fluorescence assay serum Human osteosarcoma
Matrix metalloproteinases (MMPs) are important biomarkers of various tumors. Herein, a peptide microarraybased fluorescence assay is developed for quantitatively profiling of MMP-2 activity in different matrices through the binding of the immobilized biotinylated peptides on the microarray with the fluorescein isothiocyanate (FITC) modified neutravidins. In the presence of MMP-2, the biotin moiety is released from microarray by enzymatic cleavage of peptide substrate, resulting in the decrease of fluorescence signal. The change of fluorescence intensity is correlated with MMP-2 activity. The detection limit down to 14 pg mL−1 in buffer solution is obtained by a selected peptide substrate for MMP-2 from eleven peptide substrate candidates. The cellular secreted MMP-2 activity levels of different living cells are further successfully quantitatively evaluated by the selected peptide substrate. Using mouse-bearing MG-63 human osteosarcoma as a model system, we also demonstrate that the activity of MMP-2 in serum is closely related with the cancer progression.
1. Introduction Matrix metalloproteinases (MMPs) are a kind of proteolytic enzymes with Zn2+ and Ca2+, which degrade the extracellular matrix components (ECM) and play crucial roles in physiological and pathological processes, such as normal tissue remodeling, embryogenesis, angiogenesis, wound healing, arthritis, atheroma, tissue ulceration, and cancer [1–5]. In particular, MMPs are demonstrated as useful diagnostic and prognostic biomarkers for cancer because the up-regulation of various MMPs, including MMP-1, -2, -3, -7, -9, -13, -14 are positively related with tumor progression in both primary tumors and metastases [6–10]. Due to their importance in physiological and pathological processes, many approaches/methods have been developed for detection of MMPs, including gelatinase zymography [11,12], enzyme-linked immunosorbent assays (ELISA) [13], surface plasmon resonance (SPR) assays [14,15], electrochemical biosensors [16], and fluorescence resonance energy transfer (FRET) assays [17,18]. Although these methods have reasonable accuracy and sensitivity, most of them require relative large sample volumes and cannot detect MMP activities in the high throughput format. Moreover, targeting MMP specifically is still a challenge because the active sites of MMP family members are
structurally similar [19,20]. Screening of specific peptide substrates for MMPs is beneficial for the detection MMPs activities precisely. With the advantage of massively parallel detection of a large number of different targets on a small scale, microarray has been considered as a revolutionized technology for biological detection and monitoring since it was invented in the early 1990s [21–23]. For example, peptide microarrays are extensively employed to screening antigens in practical samples and study the functionalities and inhibitions of enzymes [24–27]. Very recently, we have developed a series of peptide microarray-based fluorescence assay for profiling multiplexed MMPs activities in cell lysates and clinical thyroid tissues [28,29]. It is found that the different clinical samples exhibit different expression patterns of MMP activities. Currently, extracts of pathological tissues are normally used for analysis of tumor-associated MMPs activities [30–32]. There are few examples of monitoring MMPs activities in serum samples. Although tissue testing is a golden standard for clinical diagnosis of tumor, it is difficult to obtain tissue samples under certain conditions, such as tumorlets and brain metastasis. In addition, tissue biopsies may increase the risk of tumor metastasis. Compared to tissue biopsy, blood biopsy offers the possibility with minimize invasiveness to characterize the characteristic molecular information on tumor and monitor the
⁎ Corresponding author at: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, PR China. E-mail address: [email protected] (Z. Wang).
https://doi.org/10.1016/j.snb.2019.127320 Received 8 August 2019; Received in revised form 9 October 2019; Accepted 21 October 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Minghong Jian, et al., Sensors & Actuators: B. Chemical, https://doi.org/10.1016/j.snb.2019.127320
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2.3. Determination of MMP-2 activity in buffer
progression of cancer through real-time dynamic monitoring the expression levels of biomarkers in the blood samples of patients [33]. Therefore, MMP activity-based liquid biopsy in the blood draw has great potential in the routine diagnosis and prognosis of tumors. Herein, we have developed a peptide microarray-based fluorescence assay for profiling MMP-2 activity with high sensitivity in various biological samples, including cell culture medium, tissue extract and serum by a selected peptide substrate. Using mouse-bearing MG-63 human osteosarcoma as a typical model, the as-proposed peptide microarray-based fluorescence assay is used to robustly quantify the differential activities of MMP-2 in the serum from mice with different cancer progression. It is demonstrated that the activity of MMP-2 is associated with tumor progression.
The 10 nM proMMP-2 and 100 nM proMMP-7 were activated with 1 mM APMA in TCNB buffer at 37 °C for 1 h and 2 h, respectively. Subsequently, 30 μL various concentrations of pure MMP-2 and MMP-7 in TCNB buffer were added to each subarray and incubated at 37 °C for 4 h. The subarray treated with TCNB buffer was used as a blank control. Then, the slides were washed with washing buffer 2 for 5 min (3 × 30 mL), and Milli-Q water for 3 min (3 × 30 mL), respectively. After dried by centrifugation, 30 μL 3 μM Neutravidin-FITC in probe buffer (pH 7.5, 50 mM PB, 0.15 M NaCl, 0.1% Tween-20 (v/v) and 1% BSA (w/v)) were added to each subarray and incubated at 30 °C under dark for 1 h. After PTFE masks were removed, the glass slides were washed with PBS (pH 7.5, 0.05 M PB, 0.15 M NaCl, supplemented with 0.1% Tween-20 (v/v)) for 5 min (3 × 30 mL), and Milli-Q water for 3 min (3 × 30 mL), and dried via centrifugation before scanning, respectively. To investigate the specificity of as-proposed method for detection of MMP-2, 30 μL different enzymes (caspase 3, cathepsin B, pronase, λexo and proteinase K) in reaction buffer were added to each subarray and incubated for 4 h, respectively. The subarrays were then treated as previously described.
2. Experimental section 2.1. Reagents Recombinant human matrix metalloproteinase 2 (proMMP-2) and caspase 3 were purchased from Sino Biological Inc. (Beijing, China). Recombinant human matrix metalloproteinase 7 (proMMP-7) and cathepsin B were purchased from R&D Systems Inc. (Minneapolis, USA). Pronase from streptomyces griseus and proteinase K were purchased from Sigma-Aldrich Co. (St Louis, USA). λ exonuclease (λexo) was acquired from New England Biolabs Ltd. (Hitchin, UK). Neutravidin-FITC was acquired from Thermo Fisher Scientific Co., Ltd. (China). (4-aminophenyl) mercuric acetate (APMA) was obtained by GenMed Medical Science and Technology Ltd. (Shanghai, China). The peptide substrates (as shown in Table S1) were synthesized by Synpeptide Co., Ltd. (Shanghai, China). Bovine Serum Albumin (BSA) was acquired from GEN-VIEW Scientific Inc. (USA). Dulbecco modified eagle medium (DMEM) were supplied by Beijing Dingguo Biotechnology Ltd. (Beijing, China). McCoy’s 5A culture medium was purchased from Jiangsu KeyGEN BioTECH Co. Ltd. (Jiangsu, China). Fetal bovine serum (FBS) and trypsin-EDTA cell detaching kit were purchased from Gibco Co. (New York, USA). Human serum was obtained from First Hospital of Jilin University (Changchun, China). Gelatin-sepharose 4B was received from Solarbio Science & Technology Co., Ltd. (Beijing, China). MMP-2 activity quantification kit and Bradford protein concentration quantification kit were acquired from Shanghai Harling Biotechnology Co., Ltd. (Beijing, China). 3D aldehyde glass slides and polytetrafluoroethylene (PTFE) masks were supplied by CapitalBio Ltd. (Beijing, China). NCM460, HepG2, SW620, HCT116, HeLa and MG-63 cells were acquired from Chinese Academy of Sciences Cell Bank (Shanghai, China). All other reagents were of analytical grade. Milli-Q water (18.2 MΩ⋅cm) was used in all experiments.
2.4. Data acquisition and analysis The fluorescence signals were obtained with a LuxScan-10 K fluorescence microarray scanner (CapitalBio Ltd., Beijing, China), and background fluorescence signals were subtracted. The average value and standard deviation of each sample was calculated from six repeated signal spots. The relative change of fluorescence intensity (ΔFR) was used to represent the MMP-2 activity, which was calculated by the following formula: ΔFR = (F0–F)/F0 × 100%, where F0 and F indicate the average fluorescence signal before and after MMP-2 cleavage, respectively. The detection limit was estimated as 3 times the standard deviation of ΔFR of control samples. 2.5. Detection of cellular MMPs activities NCM460, HepG2, SW620, HCT116, HeLa and MG-63 cells were seeded in 48-well plates at a density of 5000 cells per well and grew in complete medium (fresh culture medium containing 10% fetal bovine serum (FBS) and 100 U mL−1 penicillin-streptomycin) in humidified air with CO2 at 37 °C for 24 h. NCM460, SW620 and HCT116 were cultured with McCoy’s 5A. HeLa, MG-63, and HepG2 were cultured with DMEM. The cells were washed with PBS, and incubated with fresh serum-free medium for 12 h. The culture medium was discharged. After washed with PBS, the starved cells were incubated with serum-free medium for another 24 h. Subsequently, the culture medium was collected and centrifuged (1000 rpm) at 4 °C for 10 min. Finally, the obtained supernatant was adjusted to pH 7.5 and 30 μL samples were added to each subarray for MMP-2 activity detection. The subsequent experimental procedure was as same as described to detect MMP-2 activity in buffer. For comparison, the cellular secreted MMP-2 activity in concentrated (10 times) sample was also detected by the commercial MMP-2 activity quantification kit.
2.2. Fabrication of peptide microarray The different concentrations of peptide substrates in spotting buffer (pH 8.5, 0.3 M PB, 0.2 M NaCl, containing 20 μg mL−1 BSA and 35% (v/ v) glycerol) were spotted on commercial 3D aldehyde glass slides with a SmartArrayer 136 system using a standard contact printing procedure (Capital Bio, Beijing, China). After an overnight incubation under vacuum at 30 °C, the peptide microarrays were washed with washing buffer 1 (pH 7.5, 50 mM PB, 1% (w/v) BSA) for 10 min twice, and then incubated in blocking buffer (pH 7.5, 50 mM PB, 0.15 M NaCl, 1% (w/ v) BSA and 1% (v/v) ethanolamine) at 30 °C for 1 h to block the residual aldehyde groups on the slides. After the blocking process, the slides were successively washed with washing buffer 2 (pH 7.5, 20 mM Tris, 0.15 M NaCl with 0.1% Triton X-100) for 10 min (2 × 30 mL), and then TCNB buffer (pH 7.5, 50 mM Tris, 10 mM CaCl2, 150 mM NaCl and 0.05% Brij-35) for 10 min (2 × 30 mL), respectively. Finally, the peptide microarray was dried by centrifugation at 2000 rpm for 1 min, and divided into 12 individual subarrays using a PTFE mask for further study of the MMPs activities.
2.6. Detection of MMP-2 activity in serum and tissue All the animal experiments were approved by the Regional Ethics Committee of Jilin University, and complied with the guidelines for care and use of laboratory animals. Male NOD/SCID mice (18–20 g bodyweight) were obtained from Beijing HFK Bioscience Co., Ltd. (Beijing, China), which were housed in a 12 h light/dark cycle with a controlled temperature and humidity, and fed with a chow diet and drinking tap water. After acclimatizing to the environment for one week, each NOD/SCID mouse was inoculated subcutaneously with MG2
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63 cells (1.0 × 106 cell in 100 μL PBS). When the tumor grew to a certain volume, the mice were anesthetized by isoflurane (Shenzhen RWD Life Technology Co., Ltd.). The tumors and bloods of mice were collected. The leg muscles and bloods of healthy NOD/SCID mice were collected as control samples. After standing at the room temperature for 1 h, the blood samples of NOD/SCID mice were centrifuged (1500 rpm) at 4 °C for 10 min. The serum was then collected. MMPs of tissue samples were extracted and purified according to the previously reported procedure [29]. Generally, the tissues (30 mg) were cut into small pieces with scissors and homogenized with 400 μL working buffer (pH 7.6, 50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, 0.05% (w/v) Brij35, and 0.02% (w/v) NaN3) containing 1% (v/v) Triton X-100 in 1 mL Teflon-glass homogenizer on ice. The total protein content in 10 μL homogenate was calculated using the bicinchoninic acid (BCA) method. The homogenate supernatant was collected by centrifugation (12,000 rpm) at 4 °C for 15 min, and then incubated with 50 μL gelatinsepharose 4B under constant shaking (100 rpm) for 1 h. The gelatinsepharose 4B was pretreated by working buffer (3 × 100 μL). After centrifuged at 7000 rpm for 5 min, the gelatin-sepharose pellet was resuspended in 200 μL working buffer and centrifuged again. Then, the pellet was incubated with 50 μL elusion buffer (working buffer containing 10% (v/v) DMSO) under constant shaking (100 rpm) for 30 min. The mixture was centrifuged (7000 rpm) at 4 °C for 5 min, and the supernatant was diluted to suitable volume for detecting MMPs activities. The activities of MMP-2 in serum and/or tissue extracts were detected by both the peptide microarray-based fluorescence assay and commercial MMP-2 activity quantification kit as previously described, respectively.
Fig. 1. Fluorescence images (a and b) and corresponding data analysis (c) of different peptide substrates (0.1 mg mL−1) cleaved by MMP-2 and MMP-7, respectively. The concentrations of MMP-2 and MMP-7 are 250 ng mL−1 and 100 ng mL−1, respectively.
3. Results and discussion
from the literature reported different preferred MMP-2 and MMP-7 cleavage motifs because MMP-2 and MMP-7 are normally overexpressed in the various tumors [5,9,35]. The reactions of immobilized peptides on microarray with MMPs were carried out under our previously optimized experimental conditions [29,36]. As shown in Fig. 1, there are obvious differences in the enzymatic cleavage efficiencies of S0, S3, S6 and S7 by MMP-2 and MMP-7. In particular, S0 shows much higher (5.5 times) MMP-2 cleavage efficiency than MMP-7 cleavage efficiency. The result indicates that S0 has relative high specificity for MMP-2. Therefore, S0 was employed to evaluate the activity of MMP-2 in the following experiments.
3.1. Screening the specific peptide substrate for MMP-2 Scheme 1 illustrates the detection of MMP activity using peptide microarray-based fluorescence assay. Briefly, the biotin-modified peptides were immobilized on the aldehyde 3D glass slides via forming a stable five-membered-ring thiazolidine between the N-terminals of peptide substrates and aldehyde groups on the slides [34]. NeutravidinFITC can specifically reacted with the biotin group at the C-terminal of peptide substrate to produce strong fluorescence signal. When MMPs were introduced, the peptide substrates were enzymatically cleaved and the peptide fragments containing biotin were released from the slides, resulting in the decrease of the fluorescence signal. The activities of MMPs can be expressed by fluorescence intensity changes on the microarrays. For improving the detection performance of MMPs, eleven biotinmodified peptides (named as S0 to S10) containing MMP recognition sequences were randomly designed to select the substrates of MMP-2 with relatively high specificity. The recognition sequences are derived
3.2. Determination of MMP-2 activity in buffer The effect of the surface densities of S0 on the enzymatic cleavage efficiency was firstly investigated. Figure S1 illustrates that the highest enzymatic cleavage efficiency was obtained when the concentration of S0 was 0.1 mg mL−1 in the spotting solution. In the following studies, 0.1 mg mL−1 was chosen as the optimized concentration of S0 to
Scheme 1. The schematic representation of as-proposed peptide microarray-based fluorescence assay for determining MMP-2 activity. The illustration is not drawn to scale. 3
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detection are summarized in Table 1. The results are comparable to or better than those reported in the literatures [14,18,38]. For testing its specificity for MMP-2 detection, the as-proposed peptide microarray-based fluorescence assay was employed to react with different enzymes including caspase 3, cathepsin B, pronase, λexo and proteinase K. As shown in Figure S2, those enzymes show negligible cleavage efficiency on S0, indicating that the as-proposed peptide microarray-based fluorescence assay has excellent specificity for detecting MMP-2 activity. 3.3. Detection of cellular secreted MMP-2 activities To further study its performance, the activity of cellular secreted MMP-2 was detected by the peptide microarray-based fluorescence assay. Herein, six cell lines were selected including one normal cell (human colon epithelial cell (NCM460)), five cancer cells (human cervical adenocarcinoma cell (HeLa), two kinds of human colorectal cancer cells (HCT116 and SW620), human hepatoma cell (HepG2) and human osteosarcoma cell (MG-63)). All cells were seeded with an optimal density of 5000 cells per well [29]. The activity of cellular secreted MMP-2 in culture medium was detected by the peptide microarray-based fluorescence assay. The activity level of cellular secreted MMP-2 exhibits significant difference among different cell lines (as shown in Fig. 3). For instance, the activities of cellular secreted MMP-2 by tumor cells are higher than that of the normal cell (NCM460). Although both SW620 and HCT116 are derived from colorectal cancer, the activity of cellular secreted MMP-2 by SW620 is higher than that of HCT116. In addition, the activities of secreted MMP-2 by SW620, MG63 and HepG2 are higher than those of HCT116 and HeLa. The result suggest that the MMP activity is associated to pathological characteristics of tumors, such as the degree of tumor malignancy [7,39–41]. The activity levels of cellular secreted MMP-2 by different cells were also determined by commercial MMP-2 activity quantitative determination kit (as shown in the Fig. 3), which are comparable with those obtained from the peptide microarray-based fluorescence assay. Furthermore, the sensitivity of peptide microarray-based fluorescence assay is much higher than that of commercial MMP-2 activity quantitative determination kit. The result demonstrates that the peptide microarray-based fluorescence assay can be used to effectively detect MMP-2 activity in complex biological samples.
Fig. 2. Fluorescence images (a to d) and the performance of as-proposed peptide microarray-based fluorescence assay (e) for determining MMP-2 activity in TCNB buffer (a), DMEM (b), human serum (c) and DMEM supplemented with human serum (d), respectively.
fabricate peptide microarray. Various concentrations of activated MMP2 were prepared for evaluating the performance of as-proposed peptide fluorescence assay. As shown in Fig. 2, ΔFR of S0 is linearly increased with increasing the logarithm of MMP-2 concentration. For testing the practical capability of the assay, the potential interfering substances in biological samples were investigated. The various amounts of activated MMP-2 were mixed with DMEM, 5% human serum in TCNB buffer and/ or DMEM supplemented with 5% human serum. The mixtures were then applied to subarrays. It can be seen from Fig. 2 that the interfering substances have strong effect on the detection sensitivity (slope of calibration curve) of the assay because these biosubstances normally contain small amounts natural MMP inhibitors such as TIMPs (tissue inhibitors of metalloproteinases, e.g., TIMP-1 and TIMP-2) or α2-macroglobulin [35,37]. However, the interfering substances show slight effect on the detection limit and linear range. For comparison, detection sensitivities, the linear ranges, linear regression equations and limits of
3.4. Analysis of MMPs activities in serum and tissue Encouraged by the detection result of cellular secreted MMP-2, the peptide microarray-based fluorescence assay was further used to study the relationship of MMP-2 activity with tumor progression. In this case, a NOD/SCID mouse-bearing MG-63 human osteosarcoma was used as a typical model system because human osteosarcoma has high metastatic characteristics [42]. The tumor tissues and bloods of mice were collected when the tumor grew to a specific size (0 (0 means healthy mouse), 0.5 cm, 0.8 cm or 1.2 cm maximum diameter of tumor, as shown in Fig. 4a). We systematically evaluated the MMP-2 activities of the as-collected tumor tissues and blood samples by the peptide microarray-based fluorescence assay and commercial MMP-2 activity quantitative determination kit. Different dilution volume ratios (1%, 5%, 10%, 50% and 100%) of serum with TCNB buffer were applied to
Table 1 Summary of the performance of as-proposed peptide microarray-based fluorescence assay for determining MMP-2 activity in different matrices. Matrix
Detection sensitivities
linear ranges
Linear regression equations
Limits of detection
TCNB DMEM Human Serum DMEM and Human Serum
9.32438 5.92849 5.80310 4.77283
25 25 25 25
Y = 9.32438X-7.78706 Y = 5.92849X-1.87238 Y = 5.80310X-3.16496 Y = 4.77283X-0.91547
14 pg/mL 19 pg/mL 22 pg/mL 25 pg/mL
pg/mL-500 ng/mL pg/mL-500 ng/mL pg/mL-500 ng/mL pg/mL-250 ng/mL
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Fig. 3. Profiling of cellular secreted MMP-2 activity in NCM460, HepG2, SW620, HCT116, HeLa, and MG-63 cell culture medium by peptide microarraybased fluorescence assay and commercial MMP-2 activity quantitative determination kit. The insets are the corresponding fluorescence images. For peptide microarray-based fluorescence assay, the MMP-2 amount was estimated by the calibration curve of MMP-2 in DMEM (as shown in Fig. 2e). The sample amount of commercial kit is 10 times larger than that of peptide microarraybased fluorescence assay.
Fig. 5. Profiling of MMP-2 activity in tissues homogenate obtained from the mice with different stages of MG-63 human osteosarcoma by peptide microarray-based fluorescence assay and commercial MMP-2 activity quantitative determination kit. The insets are the corresponding fluorescence images. For peptide microarray-based fluorescence assay, the MMP-2 amount was estimated by the calibration curve of MMP-2 in DMEM plus human serum (as shown in Fig. 2e). The sample amount of commercial kit is 10 times larger than that of peptide microarray-based fluorescence assay.
the subarrays for investigating the effect of serum concentration on the MMP-2 activity, because the components in serum (e.g., serum albumin and cytokines) may affect the interaction of MMP-2 with the immobilized S0 on microarrays. The MMP-2 activity in serum can be detected as low as 1% diluted serum, while the maximum cleavage efficiency was achieved with 5% diluted serum (as shown in Fig. 4b). Therefore, the 5% diluted serum was used to investigate the relationship of serum MMP-2 activity with tumor progression. Given a reaction solution volume of 30 μL, this means that the MMP-2 activity in 1.5 μL serum can be detected. As expected, the serum MMP-2 activity level is increased with increasing the tumor size (as shown in Fig. 4c). The MMP-2 activity level in tumor tissue was also determined by the peptide microarray-based fluorescence assay. The MMP-2 activity level in tumor tissue is also proportional to the tumor size (as shown in Fig. 5), i.e., the MMP-2 activity is strongly associated with osteosarcoma progression, which is consistent with the previously reported results [43–45]. The consistency of MMP-2 activity in serum and tissue suggests that MMP activity-based liquid biopsy could be used to monitor the progress of tumors. Moreover, the activities of MMP-2 in serum samples and tumor tissues were also detected by commercially available MMP-2 activity quantitative determination kits, and the results are in consistent with those of the peptide microarray-based fluorescence assay. 4. Conclusions In summary, we have proposed a peptide microarray-based fluorescence assay for profiling the MMP-2 activity in different biological samples (i.e., cell lysates, serum and tissue homogenate) with a wide dynamic range and relative high sensitivity. Using MG-63 tumorbearing NOD/SCID as a model, we demonstrate that MMP-2 activity is strongly associated with the tumor progression. Although only MG-63 human osteosarcoma related MMP-2 activity is detected in this proof of principle experiment, this approach offers a possibility for the development of MMP activity-based tumor liquid biopsy in the blood draw.
Fig. 4. Profiling of MMP-2 (b) activity in different concentrations of serum in TCNB buffer obtained from the mice with different stages of MG-63 human osteosarcoma (a) by peptide microarray-based fluorescence assay. Data analysis of MMP-2 activity in 5% serum measured by peptide microarray-based fluorescence assay and commercial MMP-2 activity quantitative determination kits (c). The insets are the corresponding fluorescence images. For peptide microarray-based fluorescence assay, the MMP-2 amount was estimated by the calibration curve of MMP-2 in human serum (as shown in Fig. 2e). The sample amount of commercial kit is 10 times larger than that of peptide microarraybased fluorescence assay.
Declaration of Competing Interest The authors declare no competing financial interest. Acknowledgement The authors would like to thank the National Natural Science Foundation of China (Grant no. 21775145) for financial support. 5
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Appendix A. Supplementary data
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Minghong Jian is currently a PhD student in the University of Science and Technology of China. She is currently working on the detection of enzyme activity in biological samples using peptide microarrays. Min Su completed her PhD degree in 2014 from University of Chinese Academy of Sciences. Her current scientific interest concentrates on the detection of thrombin enzyme. Jiaxue Gao is pursuing PhD degree at the University of Chinese Academy of Sciences. She works on cancer related-miRNA detection in living cells. Zhenxin Wang is a professor at State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, since 2006. He completed his PhD degree in the Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, in 2000. His main research interests concentrate on microarrays, multimodal imaging in vivo and biomaterials.
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